The Functional And Mechanism Studies Of Golgi Membrane Protein 1 In Malignant Biological Behaviours Of Glioblastoma

BackgroundGlioblastoma multiforme(GBM,WHO IV)accounts for 45-50%of all primary malignant brain tumors.The median survival of GBM patients is only 12-15 months even maximal safe resection supplemented with radio-and chemo-therapy which mainly depended on Temozolomide(TMZ)were taken.Diffuse infiltration into adj acent brain parenchymal and rapid proliferation of tumor cells are important reasons which led to dismal prognosis and recurrence of GBM.Although studies have demonstrated that the unique structure of extracellular matrix in the brain,activation of adherence-related cell surface receptor and multiple signaling pathways in diffused cells are main pathological basis of GBM invasion,however,no reliable approaches can be employed to suppress these abnormal alterations.In terms of malignant proliferation,epidermal growth factor receptor(EGFR)is a representative therapeutic target.However,small molecule inhibitors targeting EGFR,such as Gefitinib and Erlotinib,are still not sufficient to prolong the progression free survival(PFS)of GBM patients significantly.The efficiency of anti-proliferation drugs for other targets,such as heat shock proteins or cyclin-dependent proteins remain to be further evaluated.Thus,we still need to explore internal mechanisms of GBM proliferation,invasion and migration,in order to change the embarrassments of GBM treatment through novel approaches and conceptions.Golgi apparatus-related proteins have been reported to participate in the mediation of GBM progression.For instance,abnormal elevated expression of Golgi phosphoprotein 3(GOLPH3)promoted proliferation,invasion,migration of GBM significantly and predicted poor prognosis of GBM patients.Golgi membrane protein 1(GOLM1),also known as GOLPH2 or GP73,is a highly-phosphorylated protein located in the cis and medial-Golgi apparatus.GOLM1 mainly participates in the modification of proteins synthesized by endoplasmic reticulum and assists Golgi apparatus to accomplish protein trafficking and secretion.In recent years,the role of GOLM1 in human cancers has been revealed gradually.Literatures reported that GOLM1 could facilitate the proliferation,invasion and migration of liver cancer cells as an oncogene and had been identified as a serum diagnostic marker of liver cancer.Furthermore,GOLM1 was closely related with progression of multiple common human cancers,including prostate cancer,melanoma,gastric cancer and esophagus cancer.However,the role of GOLM1 in GBM proliferation,invasion and migration remains unclear.Gene clustering analysis performed among large number of GBM cases in The Cancer Genome Atlas(TCGA)found that GBM could be classified into four subtypes according to characterized molecular markers,including classical,mesenchymal,proneural and neural.This study laid the foundation for individualized treatment of GBM based on molecular subtypes.As a molecular marker of proneural GBM,platelet-derived growth factor receptor a(PDGFRa)and its ligand platelet-derived growth factor subunit A(PDGFA)can promote the malignant progression of GBM through both autocrine and paracrine ways.A study performed in liver cancer cell line Huh7 identified that PDGF could stimulate the expression of GOLM1,while GOLM1 existed as a key protein to participate in regulation of molecules downstream to PDGF.Nevertheless,interrelationships between GOLM1 and PDGFA/PDGFRa in GBM and potential therapeutic applications of GOLM1 in proneural GBM remains unrevealed.Based on the above research background,this study aims to investigate roles and underlying mechanisms of GOLM1 in GBM,shed a light on the relationship between GOLM1 and PDGFA/PDGFRa signaling pathway,provide novel theoretical evidence for the treatment of GBM through qRT-PCR,Western blot,immunohistochemistry,immunofluorescence,lentivirus-mediated transfection,3D tumor spheroid invasion assay,cancer-related phospho-kinase antibody array,intercranial and subcutaneous nude mice tumor model etc.Part ?:The expression and subcellular localization of GOLM1 in gliomasObjectivesWe aim to detect the expression level of GOLM1 in normal brain tissues,glioma tissues,normal human astrocyte(NHA),GBM cell lines and analyze the distribution of GOLM1 in GBM cells preliminarily.Methods1.qRT-PCR,immunohistochemistry and Western blot were used to analyze the expression of GOLM1 in normal brain tissues and different grade of glioma tissues.2.qRT-PCR and Western blot were used to detect the expression of GOLM1 in NHA and GBM cell lines(U87MG,U251,A172).3.Immunofluorescence and cytoskeleton staining were used to observe the distribution of GOLM1 in U87MG and U251 cells.Results1.GOLM1 was elevated in glioma tissues in pathological grade-dependent manner.The results of qRT-PCR,immunohistochemistry and Western blot showed that the expression of GOLM1 in normal brain tissues was generally low,and it increased from low grade glioma tissues(WHO ?)to high grade glioma tissues(WHO ?-?).2.GOLM1 was intensively expressed perinuclear of GBM cells.The results of qRT-PCR and Western blot demonstrated that U251 and A172 cells had apparently higher expression of GOLM1 than NHA and U87MG cells.Immunofluorescence and cytoskeleton staining showed that GOLM1 protein was mainly located in perinuclear cytoplasm in U87MG and U251 cells,which was consistent with the distribution of Golgi apparatus in multiple cell types.Conclusions1.GOLM1 was highly expressed in glioma tissues related to pathological grade.2.GOLM1 was expressed in perinuclear cytoplasm in GBM cells.Part ?:Roles and mechanisms of GOLM1 in the proliferation,invasion and migration of glioblastomaObjectivesWe aim to investigate the function and underlying mechanisms of GOLM1 in GBM proliferation,invasion and migration.Methods1.EdU assay,Cell Counting Kit-8(CCK-8)cell viability assay and clone-forming assay were used to detect changes of GBM cells' proliferation after knockdown or overexpression of GOLM1.2.Transwell assays were used to detect changes of GBM cells' invasion and migration after knockdown or overexpression of GOLM1.3.CCK-8 and 3D tumor spheroid invasion assays were used to detect changes of P3#GBM primary cells' proliferation and invasion after knockdown of GOLM1.4.Orthotopic and subcutaneous nude mice tumor xenografts were used to verify the significance of GOLM1 in GBM proliferation and invasion.5.Phospho-kinase antibody array and Western blot were employed to explore the molecular pathway of GOLM1-promoted GBM proliferation,invasion and migration.6.CCK-8,EdU and Transwell assays were used to explore the significance of AKT phosphorylation in GOLM1-promoted malignant behaviours of GBM.Results1.Knockdown of GOLM1 inhibited proliferation,invasion and migration of U251 and A172 cells.The results of qRT-PCR and Western blot demonstrated that lentivirus integrated shRNA of GOLM1 down-regulated GOLM1 in high efficiencies in U251 and A172 cells.The results of EdU,CCK-8 and clone-forming assays identified that GOLM1 knockdown decreased proliferation of U251 and A172 cells dramatically.Besides,we observed that GOLM1 knockdown led to morphology changes of U251 and A172 cells from long-spindle shape to short round shape under optical microscope,which implied possible changes of cell mobility.The results of Transwell assays confirmed that GOLMI knockdown could attenuate invasion and migration of U251 and A172 cells significantly.2.Knockdown of GOLM1 inhibited the progression of U251-initiated GBM.Studies in orthotopic models found that survival time of mice in GOLM1 knockdown group was prolonged significantly,while tumor volume and the number of micro satellite lesions were lower compared with control group.Immunofluorescence of human origin cells' marker TRA1-85/CD147 found that all of the invasive foci in normal brain tissues were initiated from the tumor core,which elucidated that knockdown of GOLM1 inhibited tumor cells' infiltration from the tumor core to adjacent brain parenchyma.Compared with control group,the expression of Ki-67 in GOLM1 knockdown group was significantly lower than control group as shown by the results of immunohistochemistry.Besides,we observed the decrease in volume and weight of subcutaneous tumors in GOLM1 knockdown group,which preliminarily demonstrated that GOLM1 knockdown could inhibit the progression of U251-initiated GBM.3.Knockdown of GOLM1 inhibited proliferation and invasion of P3#GBM cells.Knockdown of GOLM1 dramatically attenuated proliferation and invasion of P3#GBM cells,which was demonstrated by results of CCK-8 and 3D tumor spheroid invasion assays.In orthotopic models,the post-surgery survival time of mice in GOLM1 knockdown group was significantly prolonged,while tumor growth and invasion were evidently suppressed compared to control group.In subcutaneous models,both of tumor volume and weight in GOLM1 knockdown group were generally lower than those in control group,which indicated that knockdown of GOLM1 inhibited the progression of P3#GBM-initiated GBM significantly.4.Overexpression of GOLMl promoted proliferation,invasion and migration of U87MG cells.The results of qRT-PCR and Western blot demonstrated that lentivirus integrated overexpression plasmid of GOLM1 was sufficient to up-regulate GOLM1 in U87MG cells significantly.The results of EdU,CCK-8 and clone-forming assays indicated that overexpression of GOLM1 strengthened U87MG cells' proliferation significantly.Under optical microscope,GOLM1-overexpressed cells have longer spindle morphology and closer connection with adjacent cells compared with control group.Transwell assays identified that ectopic expression of GOLM1 elevated invasion and migration potential of U87MG cells significantly.In the experiments in vivo,we found that overexpression of GOLM1 could reduce the survival time of tumor-bearing mice significantly and boost tumor growth and invasion.In consistent with this,both of volume and weight of subcutaneous tumors in GOLM1 overexpression group was significantly higher than those in control group,which demonstrated that up-regulation of GOLM1 could accelerate the progression of U87MG-initiated GBM.5.GOLM1 promoted GBM proliferation,invasion and migration through—activation of AKT.The results of cancer-related phospho-kinase antibody array indicated that knockdown of GOLM1 could evoke phosphorylation changes of multiple cancer-associated kinases.We speculated that phosphorylation of AKT was the driving force of GOLM1-promoted GBM progression based on the importance among changed kinases in GBM and further analysis by Western blot.The expression level of proteins downstream to AKT was consistent with GOLM1.A specific inhibitor for AKT phosphorylation,MK-2206,could reverse GOLM1 overexpression-induced GBM proliferation,invasion and migration in a large extent,which indicated that AKT activation played crintical roles in oncogenic processes of GOLM1.ConclusionsGOLM1 can activate AKT to facilitate GBM proliferation,invasion and migration.Part ?:Interrelationships between GOLM1 and PDGFA/PDGFR?signaling pathway in glioblastomaObjectivesWe aim to reveal correlations between GOLM1 and PDGFR?,investigate the regulation of PDGFA/PDGFR? onto GOLM1,excavate the potential role of GOLM1 in PDGFA/PDGFR?-induced activation of downstream signaling pathways and following malignant behaviours of GBM.Methods1.TCGA and Rembrandt databases were employed to study the expression of GOLM1 in different subtype of gliomas and the correlation with PDGFR?.2.Immunohistochemistry was performed among GBM samples in our tissue bank to explore the correlation between GOLM1 and p-PDGFR?.3.qRT-PCR,immunofluorescence and Western blot were used to investigate the mediation of PDGFA/PDGFRa onto GOLM1.4.EdU,Transwell assays and Western blot were used to explore the role of GOLM1 in PDGFA/PDGFR? signaling pathway.Results1.GOLM1 was correlated with the expression and activation of PDGFRa.Analysis among different subtype of gliomas in TCGA found that GOLM1 preferentially expressed in proneural cases.Then,we discovered the positive correlation between PDGFRa and GOLM1 among glioma samples both in TCGA and Rembrandt databases.Furthermore,the results of immunohistochemistry for GOLM1 and p-PDGFR? among GBM cases in our tumor bank identified the positive relationship between GOLM1 and p-PDGFR?,which indicated that the expression of GOLM1 might associate with phosphorylation-related activation of PDGFR? and further suggested the potential link between GOLM1 and proneural GBM.2.PDGFA/PDGFR? participated in the regulation of GOLM1 expression in A172 cells.Firstly,we tested the response of U251 and A172 cells to the stimulation of exogenous PDGFA through Western blot.After treated by equal dose of PDGFA,up-regulation of p-PDGFR? in A172 cells was apparently higher than that in U251 cells.Then,we found that exogenous PDGFA could up-regulate GOLM1 in A172 cells in a dose-dependent manner both at mRNA and protein levels according to the detection by qRT-PCR,immunofluorescence and Western blot.AG1296,a specific inhibitor for auto-phosphorylation of PDGFR?,could block the induction of PDGFA on GOLM1 in a large extent which indicated that the stimulated expression of GOLM1 was depended on the activation of PDGFR?.3.GOLM1 was a key protein in PDGFA/PDGFR?-mediated malignant behaviours of GBM.Exogenous PDGFA could enhance A172 cells' proliferation,invasion and migration significantly as indicated by the results of EdU and Transwell assays,while the stimulation became relatively weak when GOLM1 was down-regulated.The results of Western blot demonstrated that knockdown of GOLM1 could partly impair the stimulation of PDGFA/PDGFR? signaling pathway to downstream effector molecules,which suggested that GOLM1 might function as a critical protein in signal transduction of PDGFA/PDGFR?.Conclusions1.GOLM1 was correlated with the expression and activation of PDGFR? in glioma tissues.2.PDGFA/PDGFR? involved to induce the expression of GOLM1 in GBM cells.3.GOLM1 was critical for the activation of downstream effectors and GBM proliferation,invasion,migration mediated by PDGFA/PDGFR?.